The term xe2x80x9cidiopathic diabetes mellitusxe2x80x9d covers a heterologous group of disorders having common symptomatic characteristics. These symptoms include an absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria and a tendency to develop arteriosclerosis, neuropathy and nephropathy. At least two major as well as several less common variants of the disease have been identified. One of the major types of diabetes is insulin-dependent diabetes mellitus (IDDM) or Type 1 diabetes which covers about 10% of patients having diabetes. The other major type of diabetes, non-insulin dependent diabetes mellitus (NIDDM) or Type 2 diabetes, represents the remaining 90% of patients having diabetes.
Absent regular insulin replacement therapy using exogenously produced insulin and/or careful monitoring of diet, diabetes patients experience a wide range of debilitating symptoms, which can progress to coma and ultimately death.
An alternative method of treating diabetes which does not require repeated administration of insulin and/or strict monitoring of diet is the transplantation of pancreatic cells or tissue from a donor to a diabetic patient. However, a major problem with pancreatic cell tissue transplantation is the shortage of human donor tissue. Only about 3,000 cadaver pancreases become available in the United States each year while about 35,000 new cases of Type 1 diabetes are diagnosed each year. Hering et al. (1999) Graft 2:12-27.
The present invention is based, in part, on the discovery that pancreatic duct and exocrine cells are capable of serving as precursor cells. It was found that by replication, mature duct and/or exocrine cells can revert to a less differentiated cell that can then redifferentiate into islet, exocrine or mature duct cells and that external signals direct the phenotypic differentiation of these cells. Thus, pancreatic duct cells can provide a source of islet cells which can be used in transplantation procedures.
Accordingly, in one aspect, the invention features a method of promoting dedifferentiation of pancreatic cells. The method includes: obtaining a population of adult or differentiated pancreatic cells; and allowing the adult or differentiated cells to proliferate, e.g., rapidly proliferate, e.g., proliferate in the presence of an agent which promotes expansion., thereby providing dedifferentiated pancreatic cells.
In a preferred embodiment, the population of adult or differentiated pancreatic cells can be: a population substantially free of islet cells, e.g., a population from which the islet cells have been removed or have been substantially removed. In a preferred embodiment, the pancreatic cells are human pancreatic cells. In a preferred embodiment, the population of cells includes: duct cells; exocrine cells; duct and exocrine cells; less than about 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% islet cells. In a preferred embodiment, the population of cells is obtained form cells remaining after islet isolation.
In a preferred embodiment, the population of cells is selected based on the ability to attach to a container, e.g., a culture flask, e.g., a non-sticky culture flask. These cells are also referred to herein as xe2x80x9cadherent cellsxe2x80x9d. In another preferred embodiment, the cells that do not attach to the container are removed from the container and cultured in another container until until the cells attach. Preferably, the cells that do not attach to the container are removed when at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more of the surface of the container has cells attached to it. Once the cells attach, they can be used in the methods of the invention. In a preferred embodiment, the adherent cells express low levels or no insulin, e.g., the cells express less than about 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, 150 ng, 100 ng, 50 ng of insulin. In a preferred embodiment, the adherent cells have: less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the insulin content of an original sample of cells obtained from a pancreas or pancreases; less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the DNA content of an original sample of cells obtained from a pancreas or pancreases.
In a preferred embodiment, the agent which promotes expansion is: a polypeptide or fragment or analog thereof which binds TGF-xcex2, e.g., a soluble TGF-xcex2 receptor; an antibody which binds TGF-xcex2; an nucleic acid which binds to TGF-xcex2 and inhibits TGF-xcex2 expression, e.g., a TGF-xcex2 antisense molecule; at least one growth factor; combinations thereof.
In a preferred embodiment, the method includes providing an agent which promotes proliferation of adult or differentiated pancreatic cells. Preferably, the agent is a growth factor or a combination of growth factors. The growth factor can be one or more of: keratinocyte growth factor (KGF); epidermal growth factor (EGF); transforming growth factors (TGF-xcex1); hepatocyte growth factor (HGF). Preferably, the growth factor is KGF.
In a preferred embodiment, the cells are allowed to proliferate by placing the cells on a substrate, e.g., a container, e.g., a plastic container, with medium containing an agent which promotes proliferation of adult or differentiated pancreatic cells, e.g., a growth factor, e.g., KGF, EGF, TGF-xcex1, and/or HGF. In a preferred embodiment, the growth factor is a growth factor which promotes the proliferation of pancreatic duct cells, e.g., rapid proliferation of pancreatic duct cells.
In a preferred embodiment, the container is: a plastic container, e.g., a plastic flask, e.g., a non-sticky plastic flask; a plastic container wherein an extracellular matrix protein has been laid down in the container, e.g., plastic container, e.g., plastic flask. In a preferred embodiment, the extracellular matrix protein is laid down by a cell, e.g., the extracellular matrix is laid down by a cancer derived cell line, e.g., a bladder carcinoma cell line, e.g., an A431 cell line. In another preferred embodiment, the extracellular matrix protein is: added to the container; is a laminin, e.g., laminin 5; is a collagen, e.g., collagen I and/or collagen IV.
In a preferred embodiment, the cells are placed on a substrate in a glucose-containing media, e.g., the glucose-containing media comprises about 4 mM, 6 mM, 8 mM, 10 mM glucose. The media can be serum free. In a preferred embodiment, nicotinamide is added to the media; insulin/transferrin/selenium (ITS) is added to the media; bovine serum albumin (BSA) is added to the media; combinations of nicotinamide, ITS and/or BSA is added to the media.
In a preferred embodiment, the population of cells is: cultured until confluent; cultured until semi-confluent; cultured until the cells form a monolayer. In a preferred embodiment, the population of cells is cultured until at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the population of cells is cultured for at least 1, 2, 3, 5, 10, 14, 18, 20, 25, 30 or more days.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; PDX-1; IPF-1; Pref-1; lack of insulin.
In another aspect, the invention features a method of obtaining pancreatic islet cells from dedifferentiated pancreatic cells. The method includes adding an extracellular matrix component to a population of dedifferentiated pancreatic cells; and culturing the cells, to thereby obtaining pancreatic islet cells.
In a preferred embodiment, the population of dedifferentiated cells includes: dedifferentiated duct cells; dedifferentiated exocrine cells; both dedifferentiated duct cells and dedifferentiated exocrine cells. In a preferred embodiment, the cells are human cells.
In a preferred embodiment, the population of cells: is a monolayer of cells; has been cultured until semi-confluent; has been cultured until confluent. In a preferred embodiment, Lp; the population of cells has been cultured until at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; IPF-1; Pref-1; lack of insulin.
In a preferred embodiment, the extracellular matrix component is one or more of: laminin, e.g., laminin 1; collagen, e.g., collagen IV; entactin; heparin sulfate proteoglycan; nidogen. In a preferred embodiment, the extracellular matrix component is a basement membrane derived substance, e.g., a basement membrane laid down by a cell, e.g., a tumor cell, e.g., an Engelbreth-Holm-Swarm (EHS) tumor cell. Preferably, the extracellular matrix component is Matrigel(trademark). In a preferred embodiment, the extracellular component further includes: one or more growth factor(s); one or more matrix metalloproteinase(s) (MMP), e.g., MMP-2, MMP-3; combinations thereof.
In a preferred embodiment, the extracellular matrix component is added by overlaying the population of dedifferentiated cells.
In a preferred embodiment, the cells are cultured for a period of at least 1, 2, 3, 5, 7, 10, 12, 14, 16, 18, 21, 25, 28, 30, 35, 40, 42, 48, 50 or more days.
In a preferred embodiment, at least a portion of the cultured cells form cultivated islet buds (CIBs), preferably cultivated human islet buds (CHIBs). Preferably, at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the cultured cells form CIBs. The term xe2x80x9cCIBsxe2x80x9d and xe2x80x9ccystsxe2x80x9d are used interchangeably herein. In a preferred embodiment, the CIBs include: islet cells, e.g., xcex1-cells, xcex2-cells, and/or xcex4-cells; hormone positive islet cells, e.g., glucagon, insulin, somatostatin and/or pancreatic peptide positive cells; duct cells; exocrine cells; combinations thereof. In a preferred embodiment, the CIBs have: increased levels of insulin expression, e.g., as compared to the dedifferentiated pancreatic cells; increased levels of glucagon expression, e.g., as compared to the dedifferentiated pancreatic cells. In a preferred embodiment, the pancreatic cells obtained have the ability to secrete insulin, e.g., the ability to secrete insulin in response to glucose.
In another aspect, the invention features a method of obtaining pancreatic islet cells. The method includes obtaining a population of adult or differentiated pancreatic cells; allowing the population of pancreatic cells to proliferate in the presence of an agent which promotes expansion, e.g., a growth factor, to obtain a dedifferentiated population of pancreatic cells; and adding an extracellular matrix component to the population of dedifferentiated pancreatic cells, to thereby obtain pancreatic islet cells.
In a preferred embodiment, the population of adult or differentiated pancreatic cells can be: a population substantially free of islet cells, e.g., a population from which the islet cells have been removed or have been substantially removed. In a preferred embodiment, the pancreatic cells are human pancreatic cells. In a preferred embodiment, the population of cells includes: duct cells; exocrine cells; duct and exocrine cells; less than about 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% islet cells. In a preferred embodiment, the population of cells is obtained from cells remaining after islet isolation.
In a preferred embodiment, the population of cells is selected based on the ability to attach to a container, e.g., a culture flask, e.g., a non-sticky culture flask. These cells are also referred to herein as xe2x80x9cadherent cellsxe2x80x9d. In another preferred embodiment, the cells that do not attach to the container are removed from the container and cultured in another container until the cells attach. Preferably, the cells that do not attach to the container are removed when at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more of the surface of the container has cells attached to it. Once the cells attach, they can be used in the methods of the invention. In a preferred embodiment, the adherent cells express low levels or no insulin, e.g., the cells express less than about 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, 150 ng, 100 ng, 50 ng of insulin. In a preferred embodiment, the adherent cells have: less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the insulin content of an original sample of cells obtained from a pancreas or pancreases; less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the DNA content of an original sample of cells obtained from a pancreas or pancreases.
In a preferred embodiment, the method includes providing an agent which promotes proliferation of adult or differentiated pancreatic cells. In a preferred embodiment, the agent which promotes expansion is: a polypeptide or fragment or analog thereof which binds TGF-xcex2, e.g., a soluble TGF-xcex2 receptor; an antibody which binds TGF-xcex2; an nucleic acid which binds to TGF-xcex2 and inhibits TGF-xcex2 expression, e.g., a TGF-xcex2 antisense molecule; at least one growth factor; combinations thereof. Preferably, the agent is a growth factor or a combination of growth factors. The growth factor can be one or more of: keratinocyte growth factor (KGF); epidermal growth factor (EGF); transforming growth factors-xcex1 (TGF-xcex1); hepatocyte growth factor (HGF). Preferably, the growth factor is KGF.
In a preferred embodiment, the cells are allowed to proliferate by placing the cells on a substrate, e.g., a container, e.g., a plastic container, with medium containing an agent which promotes proliferation of adult or differentiated pancreatic cells, e.g., a growth factor, e.g., KGF, EGF, TGF-xcex1, and/or HGF. In a preferred embodiment, the growth factor is a growth factor which promotes the proliferation of pancreatic duct cells, e.g., rapid proliferation of pancreatic duct cells.
In a preferred embodiment, the container is: a plastic container, e.g., a plastic flask, e.g., a non-sticky plastic flask; a plastic container wherein an extracellular matrix protein has been laid down in the container, e.g., plastic container, e.g., plastic flask. In a preferred embodiment, the extracellular matrix protein is laid down by a cell, e.g., the extracellular matrix is laid down by a cancer derived cell line, e.g., a bladder carcinoma cell line, e.g., an A431 cell line. In another preferred embodiment, the extracellular matrix protein is: added to the container; is a laminin, e.g., laminin 5; is a collagen, e.g., collagen I and/or collagen IV.
In a preferred embodiment, the cells are placed on a substrate in a glucose-containing media, e.g., the glucoseontaining media comprises about 4 mM, 6 mM, 8 mM, 10 mM glucose. The media can be serum free. In a preferred embodiment, nicotinamide is added to the media; insulin/transferrin/selenium (ITS) is added to the media; bovine serum albumin (BSA) is added to the media; combinations of nicotinamide, ITS and/or BSA is added to the media.
In a preferred embodiment, the population of cells is: cultured until confluent; cultured until semi-confluent; cultured until the cells form a monolayer. In a preferred embodiment, the population of cells is cultured until at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the population of cells is cultured for at least 1, 2, 3, 5, 10, 14, 18, 20, 25, 30 or more days.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; IPF-1; Pref-1; lack of insulin.
In a preferred embodiment, the population of dedifferentiated cells: is a monolayer of cells; has been cultured until semi-confluent; has been cultured until confluent.
In a preferred embodiment, the extracellular matrix component is one or more of: laminin, e.g., laminin 1; collagen, e.g., collagen IV; entactin; heparin sulfate proteoglycan; nidogen. In a preferred embodiment, the extracellular matrix component is a basement membrane derived substance, e.g., a basement membrane laid down by a cell, e.g., a tumor cell, e.g., an Engelbreth-Holm-Swarm (EHS) tumor cell. Preferably, the extracellular matrix component is Matrigel(trademark). In a preferred embodiment, the extracellular component further includes: one or more growth factor(s); one or more matrix metalloproteinase(s) (MMP), e.g., MMP-2, MMP-3; combinations thereof.
In a preferred embodiment, the extracellular matrix component is added by overlaying the population of dedifferentiated cells.
In a preferred embodiment, the cells are cultured for a period of at least 1, 2, 3, 5, 7, 10, 12, 14, 16, 18, 21, 25, 28, 30, 35, 40, 42, 48, 50 or more days.
In a preferred embodiment, at least a portion of the cultured cells form cultivated islet buds (CIBs), preferably cultivated human islet buds (CHIBs). Preferably, at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the cultured cells form CIBs. The term xe2x80x9cCIBsxe2x80x9d, xe2x80x9cspheresxe2x80x9d and xe2x80x9ccystsxe2x80x9d are used interchangeably herein. In a preferred embodiment, the CIBs include: islet cells, e.g., xcex1-cells, xcex2-cells, and/or xcex4-cells; hormone positive islet cells, e.g., glucagon, insulin, somatostatin and/or pancreatic peptide positive cells; duct cells; exocrine cells; combinations thereof. In a preferred embodiment, the CIBs have: increased levels of insulin expression, e.g., as compared to the dedifferentiated pancreatic cells; increased levels of glucagon expression, e.g., as compared to the dedifferentiated pancreatic cells. In a preferred embodiment, the pancreatic cells obtained have the ability to secrete insulin, e.g., the ability to secrete insulin in response to glucose.
In another aspect, the invention features a method of inducing dedifferentiation in adult or differentiated pancreatic cells. The method includes: providing a population of adult or differentiated pancreatic cells which include, e.g., duct and/or exocrine cells by selecting cells based on the ability to attach to a substrate, e.g., a container; culturing the cells in a rich medium, e.g., rich DMEM/F12 serum free medium (and optionally a carbon source, e.g., glucose (e.g., 8 mM), to which can be added: an agent which promotes expansion, e.g., a growth factor, and nicotinamide; and culturing till near confluence or substantial epithelial plaques, thereby providing dedifferentiated cells.
In a preferred embodiment, the population of adult or differentiated pancreatic cells can be: a population substantially free of islet cells, e.g., a population from which the islet cells have been removed or have been substantially removed. In a preferred embodiment, the pancreatic cells are human pancreatic cells. In a preferred embodiment, the population of cells includes: duct cells; exocrine cells; duct and exocrine cells; less than about 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% islet cells. In a preferred embodiment, the population of cells is obtained from cells remaining after islet isolation.
In another preferred embodiment, the cells that do not attach to the container are removed from the container and cultured until the cells attach to the flask. Preferably, the cells that do not attach to the container are removed when at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more of the surface of the container has cells attached to it. Once the cells attach, they can be used in the methods of the invention. In a preferred embodiment, the adherent cells express low levels or no insulin, e.g., the cells express less than about 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, 150 ng, 100 ng, 50 ng of insulin. In a preferred embodiment, the adherent cells have: less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the insulin content of an original sample of cells obtained from a pancreas or pancreases; less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the DNA content of an original sample of cells obtained from a pancreas or pancreases.
In a preferred embodiment, the agent which promotes expansion is: a polypeptide or fragment or analog thereof which binds TGF-xcex2, e.g., a soluble TGF-xcex2 receptor; an antibody which binds TGF-xcex2; an nucleic acid which binds to TGF-xcex2 and inhibits TGF-xcex2 expression, e.g., a TGF-xcex2 antisense molecule; at least one growth factor; combinations thereof.
In a preferred embodiment, the agent which promotes expansion is a growth factor or a combination of growth factors. The growth factor can be one or more of: keratinocyte growth factor (KGF); epidermal growth factor (EGF); transforming growth factor-xcex1 (TGF-xcex1); hepatocyte growth factor (HGF). Preferably, the growth factor is KGF. In a preferred embodiment, the growth factor is a growth factor which promotes the proliferation of pancreatic duct cells, e.g., rapid proliferation of pancreatic duct cells.
In a preferred embodiment, the container is: a plastic container, e.g., a plastic flask, e.g., a non-sticky plastic flask; a plastic container wherein an extracellular matrix protein has been laid down in the container, e.g., plastic container, e.g., plastic flask. In a preferred embodiment, the extracellular matrix protein is laid down by a cell, e.g., the extracellular matrix is laid down by a cancer derived cell line, e.g., a bladder carcinoma cell line, e.g., an A431 cell line. In another preferred embodiment, the extracellular matrix protein is: added to the container; is a laminin, e.g., laminin 5; is a collagen, e.g., collagen I and/or collagen IV.
In a preferred embodiment, the media further includes one or more of: insulin/transferrin/selenium (ITS); bovine serum albumin (BSA).
In a preferred embodiment, the cells are cultured in a rich medium with serum, e.g., rich DMEM/F12 with serum (and optionally a carbon source, e.g., glucose (e.g., 8 mM), prior to culturing the cells in a serum free rich medium.
In a preferred embodiment, the population of cells is cultured until at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the population of cells is cultured for at least 1, 2, 3, 5, 10, 14, 18, 20, 25, 30 or more days.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; IPF-1; Pref-1; lack of insulin.
In another aspect, the invention features a method of providing islet cells, e.g., alpha cells, beta cells and/or delta cells. The method includes providing a population of adult or differentiated pancreatic cells which include, e.g., duct and/or exocrine cells selected based upon the ability to attach to a substrate, e.g., a container, culturing the cells in the presence of a rich medium, e.g., rich DMEM/F12 serum free medium (and optionally a carbon source, e.g., glucose (e.g., 8 mM), to which is added: an agent which promotes expansion, e.g., a growth factor, and nicotinamide; culturing till near confluence or substantial epithelial plaques, to thereby provide dedifferentiated cells; and contacting the layer of cells with extracellular matrix, or one or more components thereof, thereby providing newly differentiated islet cells.
In a preferred embodiment, the population of adult or differentiated pancreatic cells can be: a population substantially free of islet cells, e.g., a population from which the islet cells have been removed or have been substantially removed. In a preferred embodiment, the pancreatic cells are human pancreatic cells. In a preferred embodiment, the population of cells includes: duct cells; exocrine cells; duct and exocrine cells; less than about 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% islet cells. In a preferred embodiment, the population of cells is obtained from cells remaining after islet isolation.
In another preferred embodiment, the cells that do not attach to the container are removed from the container and cultured in another container until the cells attach. Preferably, the cells that do not attach to the container are removed when at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more of the surface of the container has cells attached to it. Once the cells attach, they can be used in the methods of the invention. In a preferred embodiment, the adherent cells express low levels or no insulin, e.g., the cells express less than about 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, 150 ng, 100 ng, 50 ng of insulin. In a preferred embodiment, the adherent cells have: less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the insulin content of an original sample of cells obtained from a pancreas or pancreases; less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the DNA content of an original sample of cells obtained from a pancreas or pancreases.
In a preferred embodiment, the agent which promotes expansion is: a polypeptide or fragment or analog thereof which binds TGF-xcex2, e.g., a soluble TGF-xcex2 receptor; an antibody which binds TGF-xcex2; an nucleic acid which binds to TGF-xcex2 and inhibits TGF-xcex2 expression, e.g., a TGF-xcex2 antisense molecule; at least one growth factor; combinations thereof.
In a preferred embodiment, the agent which promotes expansion is a growth factor or a combination of growth factors. The growth factor can be one or more of: keratinocyte growth factor (KGF); epidermal growth factor (EGF); transforming growth factor-xcex1 (TGF-xcex1); hepatocyte growth factor (HGF). Preferably, the growth factor is KGF. In a preferred embodiment, the growth factor is a growth factor which promotes the proliferation of in pancreatic duct cells, e.g., rapid proliferation of pancreatic duct cells.
In a preferred embodiment, the container is: a plastic container, e.g., a plastic flask, e.g., a non-sticky plastic flask; a plastic container wherein an extracellular matrix protein has been laid down in the container, e.g., plastic container, e.g., plastic flask. In a preferred embodiment, the extracellular matrix protein is laid down by a cell, e.g., the extracellular matrix is laid down by a cancer derived cell line, e.g., a bladder carcinoma cell line, e.g., an A431 cell line. In another preferred embodiment, the extracellular matrix protein is: added to the container; is a laminin, e.g., laminin 5; is a collagen, e.g., collagen I and/or collagen IV.
In a preferred embodiment, the media further includes one or more of: insulin/transferrin/seleniurn (ITS); bovine serum albumin (BSA).
In a preferred embodiment, the cells are cultured in a rich medium with serum, e.g., rich DMEM/F12 with serum (and optionally a carbon source, e.g., glucose (e.g., 8 mM), prior to culturing the cells in a serum free rich medium.
In a preferred embodiment, the population of cells is cultured until at least about 40%, 50%, 60%, 700%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the population of cells is cultured for at least 1, 2, 3, 5, 10, 14, 18, 20, 25, 30 or more days.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; IPF-1; Pref-1; lack of insulin.
In a preferred embodiment, the layer of cells is contacted with an extracellular matrix component which is one or more of: laminin, e.g., laminin 1; collagen, e.g., collagen IV; entactin; heparin sulfate proteoglycan; nidogen. In a preferred embodiment, the extracellular matrix component is a basement membrane derived substance, e.g., a basement membrane laid down by a cell, e.g., a tumor cell, e.g., an Engelbreth-Holm-Swarm (EHS) tumor cell. Preferably, the extracellular matrix component is Matrigel(trademark). In a preferred embodiment, the extracellular component further includes: one or more growth factor(s); one or more matrix metalloproteinase(s) (MMP), e.g., MMP-2, MMP-3; combinations thereof.
In a preferred embodiment, the extracellular matrix component is added by overlaying the population of dedifferentiated cells.
In a preferred embodiment, the cells are cultured for a period of at least 1, 2, 3, 5, 7, 10, 12, 14, 16, 18, 21, 25, 28, 30, 35, 40, 42, 48, 50 or more days.
In a preferred embodiment, at least a portion of the cultured cells form cultivated islet buds (CIBs), preferably cultivated human islet buds (CHIBs). Preferably, at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the cultured cells form CIBs. The term xe2x80x9cCIBsxe2x80x9d, xe2x80x9cspheresxe2x80x9d and xe2x80x9ccystsxe2x80x9d are used interchangeably herein. In a preferred embodiment, the CIBs include: islet cells, e.g., xcex1-cells, xcex2-cells, and/or xcex4-cells; hormone positive islet cells, e.g., glucagon, insulin, somatostatin and/or pancreatic peptide positive cells; duct cells; exocrine cells; combinations thereof In a preferred embodiment, the CEBs have: increased levels of insulin expression, e.g., as compared to the dedifferentiated pancreatic cells; increased levels of glucagon expression, e.g., as compared to the dedifferentiated pancreatic cells. In a preferred embodiment, the pancreatic cells obtained have the ability to secrete insulin, e.g., the ability to secrete insulin in response to glucose.
In another aspect, the invention features a method of treating a subject, e.g., a human subject, having a disorder characterized by insufficient pancreatic islet function. The method includes administering pancreatic islet cells obtained by the methods described herein to a subject having a disorder characterized by insufficient pancreatic islet function, to thereby treat the subject.
In a preferred embodiment, the disorder is diabetes, e.g., insulin-dependent diabetes mellitus (IDDM) or non-insulin dependent diabetes mellitus (NIDDM).
In a preferred embodiment, the pancreatic cells are obtained after pancreatic islet cell isolation from a donor pancreas.
In a preferred embodiment, the pancreatic cells are obtained from the subject having the disorder.
In another aspect, the invention features a population of pancreatic cells made by any of the methods described herein.
In a preferred embodiment, the pancreatic cells are islet cells, e.g., alpha, beta and/or delta cells. Preferably, the pancreatic islets are hormone positive islet cells, e.g., glucagon, insulin, somatostatin, pancreatic peptide positive cells, and combinations thereof. In a preferred embodiment, the pancreatic islets have: increased levels of insulin expression, e.g., as compared to the dedifferentiated pancreatic cells; increased levels of glucagon expression, e.g., as compared to the dedifferentiated pancreatic cells. In a preferred embodiment, the pancreatic cells obtained have the ability to secrete insulin, e.g., the ability to secrete insulin in response to glucose.
In another preferred embodiment, the pancreatic cells are dedifferentiated pancreatic cells. Preferably, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; IPF-1; Pref-1; lack of insulin.
The term xe2x80x9cpancreatic cellxe2x80x9d as used herein refers to a cell obtained from the pancreas. Preferably, the pancreatic cell is a duct cell or an exocrine cell. A xe2x80x9cpopulation of pancreatic cellsxe2x80x9d refers to two or more cells obtained from the pancreas. The cells can be obtained from the same or different pancreases. Preferably, a population of pancreatic cells is substantially free of islet cells, e.g., the population of pancreatic cells comprises less than 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1% pancreatic islet cells.
The terms xe2x80x9cCIBsxe2x80x9d, xe2x80x9cspheresxe2x80x9d and xe2x80x9ccystsxe2x80x9d are used interchangeably herein. These terms refer to three-dimensional structures which arise from dedifferentiated pancreatic cells contacted with an extracellular matrix component. Preferably, the CIBs include one or more of: duct cells; exocrine cells; endocrine cells, e.g., cells which stain positive for insulin, glucagon, somatostatin and/or pancreatic peptide; pancreatic islet cells, xcex1-cells, xcex2-cells, and/or xcex4-cells.
The terms xe2x80x9cIPF-1xe2x80x9d and xe2x80x9cPDX-1xe2x80x9d are used interchangeably herein. Other names for xe2x80x9cIPF-1xe2x80x9d include xe2x80x9cSTF-1xe2x80x9d and xe2x80x9cIDX-1xe2x80x9d.
The term xe2x80x9csubjectxe2x80x9d includes mammals, particularly humans, susceptible to a disease characterized by insufficient insulin activity. Examples of subjects include primates, e.g., humans and monkeys.
Other features and advantages of the invention will be apparent from the following description and from the claims.